Fatty Acid Synthase Inhibitor Research Articles

Abstract 1796: Role of FASN in pancreatic neuroendocrine cancer cells survival

Abstract Introduction. Pancreatic neuroendocrine tumors (pNETs) are among the most frequently occurring neuroendocrine neoplasms. Up-regulation of fatty acid synthase (FASN) has been reported in many cancers, but the role of lipid metabolism in pNETs remains unclear. The purpose of this study was to determine whether FASN inhibition can be used as a target for pNET treatment. Methods. Immunohistochemistry and western blot analysis were used to determine protein levels of FASN in pNET tissues and cell lines. Expression of FASN was evaluated in pancreatic neuroendocrine primary and metastatic tumors and scored by a pathologist. Neutral lipid droplet levels in pNET cell lines were determined with BODIPY 493/503 staining. Clonogenic and SRB assays were used to evaluate cytotoxic effects of FASN inhibition by TVB-3664 in QGP-1 and BON pancreatic NET cell lines. Western blot analysis was used to determine levels of cleaved PARP and cyclin D1 after FASN inhibition. FASN knockdown was used to determine the effects of FASN downregulation on proliferation and lipid synthesis. Results. FASN expression was determined at the maximum scores of 6 and 5 in 98% of patient samples. BON, QGP-1 and NT-3 cell lines were stained with BODIPY 493/503; the highest levels of neutral lipid droplets were observed in the BON cell line. BON and QGP-1 cells were treated with TVB-3664 in either complete media with fatty acids (FA+) or complete media prepared with fatty acid depleted FBS (FA-). Treatment with TVB-3664 resulted in significant inhibition of BON and QGP-1 cell proliferation and cyclin D1 expression in both FA+ and FA- conditions. Depletion of extracellular FA enhanced the effect of TVB-3664 therapy on NET cells, especially in the BON cell line. FASN inhibition with TVB-3634 suppressed pNET cell proliferation and colony formation. Conclusions. Our study identified high protein levels of FASN in pNET patient samples and cell lines. Moreover, we demonstrated variability in the response of pNET cells to FASN inhibition. QGP cell viability was significantly decreased with FASN inhibition even in the presence of extracellular lipids. FASN inhibition in BON cells had only a minimal effect on cell viability and required depletion of extracellular lipids to achieve a decrease in cell viability and cyclin D1 expression. These data demonstrate that de novo lipid synthesis is involved in the pathogenesis and progression of pNETs. Importantly, our findings show that some pNETs are highly sensitive to FASN inhibition, suggesting that FASN inhibition may be considered as a treatment option in selected pNETs. Citation Format: Benjamin M. Evers, Zeta Chow, Yekaterina Zaytseva, Eun Y. Lee, Courtney M. Townsend, Tianyan Gao, B. Mark Evers, Piotr G. Rychahou. Role of FASN in pancreatic neuroendocrine cancer cells survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1796.

Abstract 438: Inhibition of de novo lipogenesis with PUFA supplementation induces ferroptosis in advanced prostate cancer

Abstract Background: Metabolic alterations are recognized as a hallmark of cancer progression. In prostate cancer (PCa), in particular, a notable lipid reprogramming occurs with increased expression levels of multiple enzymes involved in the fatty acid synthesis pathway to fulfill the metabolic and structural demands of cancer cells. Fatty acids are vital for membrane building, ATP production through B-oxidation, and storage in lipid droplets for high-energy demands. In castration-resistant prostate cancer (CRPC), the aberrant overexpression of Fatty Acid Synthase (FASN), the rate-limiting enzyme in de novo lipogenesis (DNL), results in synthesis of saturated (palmitate) and monounsaturated fatty acids (SFA and MUFA). If DNL is inhibited, polyunsaturated fatty acids (PUFAs) are taken up from the environment. These could undergo lipid peroxidation generating reactive oxygen species (ROS) and contribute to ferroptosis. We hypothesize that combining the inhibition of DNL with the addition of exogenous PUFAs could increase cell death by ferroptosis. Methods: PCa cells (LNCaP, 22Rv1 and C4-2) and human organoids were exposed to pharmacologic FASN inhibition and PUFAs for 6 days. Cell viability, lipid peroxidation, superoxide production and, protein carbonylation as a measure of intracellular ROS as well as lipidomic profiling, were assessed. Results: FASN inhibition significantly increased lipid peroxidation and intracellular ROS, with subsequent membrane potential hyperpolarization in prostate cancer cells. These effects were partially rescued by palmitate, while FASNi supplemented with PUFA potentiated protein carbonylation. Expression levels of GPX4, the main regulator of ferroptosis, were reduced after DNL inhibition and rescued with palmitate addition. Suppression of DNL combined with supplemental addition of PUFAs resulted in significant cell growth inhibition in prostate cancer cells and organoids. Finally, lipid profiling demonstrated that the suppression of DNL triggers increased acyl chain length in phospholipid and several other lipid classes. Again, this effect was significantly potentiated when combining FASN inhibition with PUFA supplementation. Conclusion: FASN inhibition produces a cascade of intracellular events, including lipid remodeling, increased ROS production and ferroptosis, all potentiated by PUFAs. These findings provide compelling evidence for the use a combined approach involving DNL inhibition and PUFA supplementation as a novel therapeutic avenue to treat advanced prostate cancer. Citation Format: Silvia Daniele Rodrigues, Isadora Ferrari Teixeira, Pier Vitale Nuzzo, Hubert Pakula, David Nanus, Massimo Loda. Inhibition of de novo lipogenesis with PUFA supplementation induces ferroptosis in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 438.

Abstract 4642: Fatty acid synthase as a novel prognostic biomarker and a potential therapeutic target in diffuse malignant peritoneal mesothelioma

Abstract Diffuse malignant peritoneal mesothelioma (DMPM) is a rare and locally aggressive tumor with a dismal prognosis. The availability of new therapies and biomarkers for patients’ selection is still an urgent need. Altered lipid metabolism, which has been reported in several tumors, has not yet been studied in DMPM. By comparatively analyzing gene expression profiles of tumors form relapsed (n=18) and non-relapsed (n=18) DMPM patients, we initially found that fatty acid synthase (FASN) was up-modulated in relapsed tumors, with a significant linear correlation between FASN expression and shorter patient relapse-free survival (RFS), suggesting a potential role of FASN in DMPM progression. The prognostic significance of FASN was further assessed in a cohort of 80 DMPM patients who underwent cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy. Our results indicated that high FASN staining was significantly associated with reduced RFS (hazard ration (HR): 1.930; p=0.014) and Overall Survival (OS) (HR: 1.854; p=0.032) as assessed by immunohistochemistry in tissues microarray samples. The prognostic value of FASN, along with Ki67 staining and patient age, was also maintained in multivariate analysis in terms of both RFS (HR: 2.240; p=0.007) and OS (HR: 2.246; p=0.014). To validate FASN as a therapeutic target, we investigated the cytotoxic effects of non-selective (orlistat) and selective (cerulenin and C75) FASN inhibitors on patient-derived DMPM cell lines, both as single agents and in combination with selinexor -a selective inhibitor of the exportin chromosome maintenance protein. FASN pharmacological inhibition in DMPM cells resulted in a significant reduction of cell proliferation, especially upon C75 exposure. In combination experiments, we observed a synergistic activity of FASN inhibitors and selinexor, as determined by Chou and Talalay method. Through western blotting and flow cytometry analysis, we gained further insight into the mechanisms by which FASN inhibitors and selinexor synergize to reduce DMPM cell proliferation. Compared to single agents, the combined treatments resulted in increased apoptosis response and cell cycle impairment, as indicated by caspase-dependent apoptosis activation and cell accumulation in the G2-phase. Overall, our results support the relevance of FASN as a prognostic biomarker for DMPM patients and underscore its potential as a novel therapeutic target. Citation Format: Valentina Doldi, Silvia Martini, Chiara Maura Ciniselli, Alessia Beretta, Marcello Deraco, Shigeki Kusamura, Dario Baratti, Paolo Gandellini, Paolo Verderio, Nadia Zaffaroni. Fatty acid synthase as a novel prognostic biomarker and a potential therapeutic target in diffuse malignant peritoneal mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4642.

Abstract 2784: Fatty acid synthase enhances stem-like properties of normal intestinal and colorectal cancer cells via an increase in notum expression and secretion

Abstract Introduction: Overexpression of lipogenic enzymes has been associated with a poor clinical outcome in colorectal cancer (CRC). Fatty acid synthase (FASN) synthesizes palmitate which can be utilized for post-translational modifications of various proteins. Notum, a palmitoleoyl-protein, prevents the downstream activation of Wnt signaling by de-palmitoylating Wnt ligands. Notum is also a CRC stem cell marker, and its expression correlates with poor prognosis in CRC. We found that upregulation of FASN is associated with an increase in Notum expression in mouse adenoma tissues. Therefore, the purpose of this study is to elucidate the mechanisms and contribution of FASN/Notum axis in CRC. Methods: Adenoma and normal intestinal organoids, established from ApcMin/VillinCre-ERT2 and Apc/VillinCre-ERT2 mouse models with hetero- and homozygous deletion of FASN. 4-hydroxytamoxifen (4-OHT) and TVB-3664 (FASN inhibitor) treatments were used to assess the effect of FASN deletion/inhibition on organoid growth and viability. LIVE/DEAD™ Viability/Cytotoxicity and Cell Titer-Glo® 3D Cell Viability Assays were used for quantitative analysis of growth. Human NOTUM/Protein notum homolog ELISA Kit was used to analyze Notum secretion. CRC cells, NTC and FASN shRNA, and control and FASN overexpression, were used for Notum analysis. Results: The TCGA data analysis shows that Notum is significantly upregulated in CRC and its expression correlates with expression of FASN in tumor tissue. Utilizing qRT-PCR and Western blot analyses, we show that downregulation of FASN leads to a decrease in expressions of active β-catenin, Notum, and other stem cell markers in transgenic mouse models. Moreover, 4-OHT-mediated inhibition of FASN results in decrease viability and size in ApcMin/FASN/VillinCre-ERT2 and Apc/FASN/VillinCre-ERT2 organoids. Consistently, shRNA-mediated deletion of FASN decreases expression of Notum in CRC cells. In contrast, overexpression of FASN increases the expression levels of active and total β-catenin, Notum, and other stem cell markers in SW480 cells. We show that normal epithelial cells exposed to CRC cell medium exhibit stem-like phenotype which is reduced when cells are exposed to medium collected from Notum shRNA knockdown cells. Moreover, the addition of recombinant Notum restores this phenotype. Conclusion: In summary, downregulation of FASN leads to a decrease in expression of Notum and is associated with morphological changes and significant decrease in viability in organoid models. Conversely, FASN overexpression upregulates Notum expression suggesting a potential cross talk between de novo lipid synthesis and Notum. Delineating the role of FASN regulation of stem-like properties via upregulation of β-catenin signaling and expression of Notum and other stem cell markers will provide the rationale for targeting the FASN/Notum axis in CRC. Citation Format: Courtney Olivia Kelson, Josiane Weber Tessmann, Daheng He, Chi Wang, Yekaterina Zaytseva. Fatty acid synthase enhances stem-like properties of normal intestinal and colorectal cancer cells via an increase in notum expression and secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2784.

Abstract 4760: Inhibition of FASN postpones development of resistance to BRAF inhibitors in colorectal cancer

Abstract Introduction: Mutations in proto-oncogene BRAF occur in about 10-15% of CRC patients and BRAFV600E is the most common. For patients who undergo BRAF-targeted therapy, resistance develops 4-6 months after treatment initiation and results in more aggressive disease. We found that development of resistance to BRAF inhibitors (BRAFi) is associated with an increase in lipid metabolism and expression of fatty acid synthase (FASN). FASN is a key enzyme of lipid synthesis overexpressed in CRC. Therefore, our hypothesis is that inhibition lipid metabolism via FASN will postpone development of resistance to BRAFi. Methods: We established CRC cells resistant to PLX8394, a novel BRAFi. To evaluate differences in parental and resistance cells, CellTiterGlo 2.0, PrestoBlue, CytoSelectTM Invasion Assay, Triglyceride Assay, Seahorse XF, and confocal microscopy were used. Combination of PLX8394 and TVB3664 or C75 (FASN inhibitors) was tested on cell viability, colony formation, and synergy studies in parental and BRAFi resistant cells. Results: The development of resistance to BRAFi promotes cellular proliferation and increases cyclin D and survivin expression in vitro and in vivo. BRAFi resistance is also associated with an increase in invasive properties and loss of E-cadherin expression. Metabolic changes include an increase in lipid metabolism, oxidative phosphorylation, and triglycerides storage. RNAseq and western blot analysis show significant upregulation of FASN in BRAFi resistant cells. Using cell viability and soft agar colony formation assays, we show that combined PLX8394 and TVB3664 treatment leads to a significantly higher decrease in cell viability and colony formation as compared to each drug alone in parental cells but not in BRAFi resistant cells. The calculation of a Bliss synergy score confirms that combination treatment with C75 and PLX8394 has synergetic effect in BRAFV600E cells. To further confirm that FASN contributes to resistance to BRAFi, we show that HT29 FASN shRNA cells are more susceptible to PLX8394 treatment as compared to control cells. Importantly, our data show that the long-term treatment with PLX8394 in combination with TVB3664 postpones development of resistance to BRAFi as compared to cells treated with PLX8394 alone. Conclusion: Our study demonstrates that resistance to BRAFi is associated with a significant increase in proliferation, metastasis, and lipid metabolism. We demonstrate that combination of FASN inhibitors and BRAFi postpones development of resistance in BRAFV600E cells. However, FASN inhibition does not sensitize cells to BRAFi in already resistance cells, suggesting that this approach cannot be used to overcome acquired resistance to BRAFi. In summary our data suggest that an addition of TVB3664 at the beginning of BRAF treatment regimen could be an efficacious treatment strategy for BRAFV600E CRC patients. Citation Format: Mariah Geisen, Josiane weber-Tessmann, Courtney Kelson, Daheng He, Chi Wang, Abu Saleh Mosa Faisal, Jill Kolesar, Yekaterina Zaytseva. Inhibition of FASN postpones development of resistance to BRAF inhibitors in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4760.

MSC-Derived Small Extracellular Vesicles Exert Cardioprotective Effect Through Reducing VLCFAs and Apoptosis in Human Cardiac Organoid IRI Model.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, accounting for 31% of all deaths globally. Myocardial ischemia-reperfusion injury (IRI), a common complication of CVDs, is a major cause of mortality and morbidity. Studies have shown efficacious use of mesenchymal stem cells-derived small extracellular vesicles (MSCs-EVs) to mitigate IRI in animals, but few research has been done on human-related models. In this study, human embryonic stem cell-derived chambered cardiac organoid (CCO) was used as a model system to study the effects of MSC-EVs on myocardial IRI. The results revealed that MSC-EVs treatment reduced apoptosis and improved contraction resumption of the CCOs. Metabolomics analysis showed that this effect could be attributed to EVs' ability to prevent the accumulation of unsaturated very long-chain fatty acids (VLCFAs). This was corroborated when inhibition of fatty acid synthase, which was reported to reduce VLCFAs, produced a similar protective effect to EVs. Overall, this study uncovered the mechanistic role of MSC-EVs in mitigating IRI that involves preventing the accumulation of unsaturated VLCFA, decreasing cell death, and improving contraction resumption in CCOs.

Fatty Acid Synthase (FASN): A Patent Review Since 2016-Present.

Fatty acid synthase (FASN), is a key metabolic enzyme involved in fatty acid biosynthesis and is an essential target for multiple disease progressions like cancer, obesity, NAFLD, etc. Aberrant expression of FASN is associated with deregulated energy metabolism of cells in these diseases. This article provides a summary of the most recent developments in the discovery of novel FASN inhibitors with potential therapeutic uses in cancer, obesity, and other metabolic disorders such as nonalcoholic fatty liver disease from 2016 to the present. The recently published patent applications and forthcoming clinical data of FASN inhibitors from both academia and the pharma industries are also highlighted in this study. The implication of FASN in multiple diseases has provided an impetus for developing novel inhibitors by both pharma companies and academia. Critical analysis of the patent literature reveals the exploration of diverse molecular scaffolds to identify potential FASN inhibitors that target the different catalytic domains of the enzyme. In spite of these multifaceted efforts, only one molecule, TVB-2640, has reached phase II trials for nonalcoholic steatohepatitis (NASH) and many malignancies. However, thecombined efforts of pharma companies to produce several FASN inhibitors might facilitate the clinical translation of this unique class of inhibitors. Nevertheless, concerted efforts towards developing multiple FASN inhibitors by pharma companies might facilitate the clinical translation of this novel class of inhibitors.

A phase I, open-label, dose-finding study of fatty acid synthase (FASN) inhibitor TVB-2640 administered in combination with enzalutamide (Enza) in men with metastatic castration-resistant prostate cancer (mCRPC).

TPS248 Background: Recent studies have shown that targeting lipid synthesis is a novel therapeutic strategy to overcome androgen resistance in mCRPC. TVB-2640 is a first-in-class FASN inhibitor, a key enzyme in de novo lipogenesis that is overexpressed and androgen-regulated in CRPC. FASN promotes the synthesis of palmitate that can be elongated and desaturated to produce saturated fatty acids (SFAs) and monounsaturated FA (MUFAs) that can support membrane synthesis and energy production, protect against free radicals and chemotherapeutics, and regulate post-translational modifications of major oncogenic pathways. Our preliminary in vitro and in vivo data show that TVB-2640 co-administered with enza to CRPC cells significantly reduced cell growth, increased apoptosis, and promoted cell cycle arrest, as well as down-regulating full-length androgen receptor (AR) and its splice variant, AR-V7. Methods: This is an open-label, single-arm, Phase I dose-escalation study that enrolls pts with mCRPC who are candidates to receive enza. The primary objective is to determine the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) of TVB-2640 in combination with enza. Secondary objectives include safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity. Exploratory objectives include the effects of FASN inhibition on metabolites, genetics, and lipids in human blood and tumor tissue, to identify potential predictive and prognostic markers of response. Pts with a confirmed histological or cytological diagnosis of PC, evidence of metastatic PC on imaging (bone scan and/or CT/MRI scan), serum testosterone <50 ng/dl, who had progressed on androgen-depletion therapy (ADT), with documented progressive metastatic castration-resistant prostate cancer (mCRPC) based on Prostate Cancer Working Group 3 (PCWG3) criteria a are eligible. Enrolled pts will receive enza at a daily dose of 160 mg for 36 days to reach a steady state, followed by the addition of oral TVB-2640 starting at 100 mg/daily. The dose escalation will follow the Bayesian optimal interval (BOIN) design with additional dose levels of 150 mg, 200 mg, 250 mg, and 300 mg daily. The maximum sample size for this study is 30 pts, with a target dose-limiting toxicity (DLT) rate of 25% or less and a maximum of 15 pts at any given dose level. This is the first clinical trial to evaluate the targeting of the AR pathway through inhibition of lipid synthesis as a new approach to treating mCRPC. Enrollment of Cohort 1 began in August 2023. Clinical trial information: NCT05743621 .

Inhibition of palmitoyltransferase ZDHHC12 sensitizes ovarian cancer cells to cisplatin through ROS-mediated mechanisms.

Platinum-based therapies have revolutionized the treatment of high-grade serous ovarian cancer (HGSOC). However, high rates of disease recurrence and progression remain a major clinical concern. Impaired mitochondrial function and dysregulated reactive oxygen species (ROS), hallmarks of cancer, hold potential as therapeutic targets for selectively sensitizing cisplatin treatment. Here, we uncover an oncogenic role of the palmitoyltransferase ZDHHC12 in regulating mitochondrial function and ROS homeostasis in HGSOC cells. Analysis of The Cancer Genome Atlas (TCGA) ovarian cancer data revealed significantly elevated ZDHHC12 expression, demonstrating the strongest positive association with ROS pathways among all ZDHHC enzymes. Transcriptomic analysis of independent ovarian cancer datasets and the SNU119 cell model corroborated this association, highlighting a strong link between ZDHHC12 expression and signature pathways involving mitochondrial oxidative metabolism and ROS regulation. Knockdown of ZDHHC12 disrupted this association, leading to increased cellular complexity, ATP levels, mitochondrial activity, and both mitochondrial and cellular ROS. This dysregulation, achieved by the siRNA knockdown of ZDHHC12 or treatment with the general palmitoylation inhibitor 2BP or the fatty acid synthase inhibitor C75, significantly enhanced cisplatin cytotoxicity in 2D and 3D spheroid models of HGSOC through ROS-mediated mechanisms. Markedly, ZDHHC12 inhibition significantly augmented the anti-tumor activity of cisplatin in an ovarian cancer xenograft tumor model, as well as in an ascites-derived organoid line of platinum-resistant ovarian cancer. Our data suggest the potential of ZDHHC12 as a promising target to improve the outcome of HGSOCs in response to platinum-based chemotherapy.

Non-alcoholic fatty liver disease promotes liver metastasis of colorectal cancer via fatty acid synthase dependent EGFR palmitoylation

Liver metastasis is the major reason for most of colorectal cancer (CRC) related deaths. Accumulating evidence indicates that CRC patients with non-alcoholic fatty liver disease (NAFLD) are at a greater risk of developing liver metastasis. With the growing prevalence of NAFLD, a better understanding of the molecular mechanism in NAFLD-driven CRC liver metastasis is needed. In this study, we demonstrated that NAFLD facilitated CRC liver metastasis as a metabolic disorder and promoted the stemness of metastatic CRC cells for their colonization and outgrowth in hepatic niches. Metabolically, the lipid-rich microenvironment in NAFLD activated de novo palmitate biosynthesis in metastatic CRC cells via upregulating fatty acid synthase (FASN). Moreover, increased intracellular palmitate bioavailability promoted EGFR palmitoylation to enhance its protein stability and plasma membrane localization. Furthermore, we demonstrated that the FDA-approved FASN inhibitor orlistat could reduce NAFLD-activated endogenous palmitate production, thus inhibiting palmitoylation of EGFR to suppress CRC cell stemness and restrict liver metastasis in synergy with conventional chemotherapy. These findings reveal that the NAFLD metabolic microenvironment boosts endogenous palmitate biosynthesis in metastatic CRC cells and promotes cell stemness via EGFR palmitoylation, and FASN inhibitor orlistat could be a candidate adjuvant drug to suppress liver metastasis in CRC patients with NAFLD.

Fatty acid synthase (FASN) signalome: A molecular guide for precision oncology.

The initial excitement generated more than two decades ago by the discovery of drugs targeting fatty acid synthase (FASN)-catalyzed de novo lipogenesis for cancer therapy was short-lived. However, the advent of the first clinical-grade FASN inhibitor (TVB-2640; denifanstat), which is currently being studied in various phase II trials, and the exciting advances in understanding the FASN signalome are fueling a renewed interest in FASN-targeted strategies for the treatment and prevention of cancer. Here, we provide a detailed overview of how FASN can drive phenotypic plasticity and cell fate decisions, mitochondrial regulation of cell death, immune escape and organ-specific metastatic potential. We then present a variety of FASN-targeted therapeutic approaches that address the major challenges facing FASN therapy. These include limitations of current FASN inhibitors and the lack of precision tools to maximize the therapeutic potential of FASN inhibitors in the clinic. Rethinking the role of FASN as a signal transducer in cancer pathogenesis may provide molecularly driven strategies to optimize FASN as a long-awaited target for cancer therapeutics.

Fabrication of Nanocrystals for Enhanced Distribution of a Fatty Acid Synthase Inhibitor (Orlistat) as a Promising Method to Relieve Solid Ehrlich Carcinoma-Induced Hepatic Damage in Mice.

Orlistat (ORL) is an effective irreversible inhibitor of the lipase enzyme, and it possesses anticancer effects and limited aqueous solubility. This study was designed to improve the aqueous solubility, oral absorption, and tissue distribution of ORL via the formulation of nanocrystals (NCs). ORL-NC was prepared using the liquid antisolvent precipitation method (bottom-up technology), and it demonstrated significantly improved solubility compared with that of the blank crystals (ORL-BCs) and untreated ORL powder. The biodistribution and relative bioavailability of ORL-NC were investigated via the radiolabeling technique using Technetium-99m (99mTc). Female Swiss albino mice were used to examine the antitumor activity of ORL-NC against solid Ehrlich carcinoma (SEC)-induced hepatic damage in mice. The prepared NCs improved ORL's solubility, bioavailability, and tissue distribution, with evidence of 258.70% relative bioavailability. In the in vivo study, the ORL-NC treatment caused a reduction in all tested liver functions (total and direct bilirubin, AST, ALT, and ALP) and improved modifications in liver sections that were marked using hematoxylin and eosin staining (H&E) and immunohistochemical staining (Ki-67 and ER-α) compared with untreated SEC mice. The developed ORL-NC could be considered a promising formulation approach to enhance the oral absorption tissue distribution of ORL and suppress the liver damage caused by SEC.

Fatty acid synthesis suppresses dietary polyunsaturated fatty acid use

Dietary polyunsaturated fatty acids (PUFA) are increasingly recognized for their health benefits, whereas a high production of endogenous fatty acids – a process called de novo lipogenesis (DNL) - is closely linked to metabolic diseases. Determinants of PUFA incorporation into complex lipids are insufficiently understood and may influence the onset and progression of metabolic diseases. Here we show that fatty acid synthase (FASN), the key enzyme of DNL, critically determines the use of dietary PUFA in mice and humans. Moreover, the combination of FASN inhibition and PUFA-supplementation decreases liver triacylglycerols (TAG) in mice fed with high-fat diet. Mechanistically, FASN inhibition causes higher PUFA uptake via the lysophosphatidylcholine transporter MFSD2A, and a diacylglycerol O-acyltransferase 2 (DGAT2)-dependent incorporation of PUFA into TAG. Overall, the outcome of PUFA supplementation may depend on the degree of endogenous DNL and combining PUFA supplementation and FASN inhibition might be a promising approach to target metabolic disease.

The Effect of Nano-Encapsulated Orlistat on Prostate Cancer: An In vitro Study

Background: The targeted inhibition of fatty acid synthase (FASN) by Orlistat, a potent FASN inhibitor, has been shown to block tumor proliferation and induce apoptosis in cultured tumor cells. Since Orlistat is insoluble, its solubility in blood circulation is limited. Cancer nanotherapeutics are rapidly progressing and are being implemented to solve several limitations of conventional drug delivery systems. Aim: In this in vitro study, we aimed to overcome the limitation of orlistat changes by designing a nanoparticle and investigating its selective cytotoxic effects on cancer cells. Method: To investigate the effects of encapsulated Orlistat on prostate cancer, we measured apoptosis and the distribution of cells via flow cytometry, confocal microscope, and Western blot. Results: There is an improvement in the solubility of Orlistat with the effect of nanoparticles; it was observed that Orlistat decreased the expression of FASN and caused dose-dependent cytotoxicity with Caspase-3, Bax, and PARP activation in PC-3 cells, while it did not have a significant effect on normal prostate epithelial cells. Conclusion: The present results indicate that the limitation of Orlistat can be overcome by assembling a nano-micellar formulation that can induce apoptotic death in the PC-3 cell line but normal prostate epithelial cells.

The potential of several compounds in Peperomia pellucida as a solution to rifampicin resistance in tuberculosis disease

Background: Tuberculosis is a lung disease caused by Mycobacterium tuberculosis. In 2018, the incidence of tuberculosis in Indonesia was 1,017,290. The number of cases was accompanied by an increase in the number of resistances to first-line antibiotics (rifampicin) used due to patient non-compliance as well as the long duration of treatment. The increase in resistance around the world is always increasing every year by about 2%. Therefore, we need another alternative to existing plants in Indonesia that has the potential to be a solution to the problem and overcome resistance to these treatments.Objective: This review was conducted to find out the potential of Peperomia pellucida as an antituberculosis drug that can be a solution to rifampicin resistance in tuberculosis disease. Results: The results of the literature review showed that the Peperomia pellucida or suruhan plants have antibacterial potential against Mycobacterium tuberculosis because it contains a class of alkaloid compounds that function to inhibit deprenyl-phosphoryl-β-D-ribose-2 oxidase (an enzyme responsible for the biosynthesis of arabinogalactan in the cell walls of Mycobacterium tuberculosis), a flavonoid that has activity inhibition of fatty acid synthase II (FAS-II) enzymes, tannins that can precipitate proteins in Mycobacterium tuberculosis, and triterpenoids that have an activation mechanism of intracellular killing cascades in host cells during tuberculosis bacterial infection.Conclusion: Peperomia pellucida or suruhan plants have the potential to be further investigated as a solution to overcome rifampicin resistance in tuberculosis disease.Keywords: Mycobacterium tuberculosis, rifampicin resistance, Peperomia pellucida

Computational Screening of Potential Fatty Acid Synthase Inhibitors as Broad-Spectrum Antiviral Agents

Infectious diseases caused by viruses are one of the major diseases that seriously endanger global health. The development of broad-spectrum antiviral drugs for effective control of emerging and recurrent viral infectious diseases is urgently needed. Fatty acid synthase (FAS) has been reported as a valuable target for developing broad-spectrum antiviral drugs. In this study, we aimed to screen potent FAS inhibitors as broad-spectrum antiviral agents through computational approach. A pharmacophore model was generated with crystal structure of FAS in complex with Orlistat using Pharmit server, and 2,215 compounds were screened from ZINC15 database against the pharmacophore model. Next, 12 compounds exhibited more affinity to FAS than the control compound (Orlistat) were screened using molecular docking. The top three compounds (ZINC6146291, ZINC8925941 and ZINC3257427), which showed docking affinity to FAS with −8.19 kcal/mol, −8.14 kcal/mol and −8.07 kcal/mol, respectively, were subjected to 200 ns molecular dynamics simulation to evaluate the stability of docked complexes and their interaction mechanisms. The simulation trajectory analysis showed the stable conformation observed in FAS bound of the compounds and the screened compounds were firmly bound of the active sites of FAS throughout molecular dynamics simulation. The screened FAS candidate inhibitors can be used as broad-spectrum antiviral agents for in vitro-in vivo evaluations.

Lipidomics Profiling Reveals Differential Alterations after FAS Inhibition in 3D Colon Cancer Cell Culture Models.

Cancerous cells synthesize most of their lipids de novo to keep up with their rapid growth and proliferation. Fatty acid synthase (FAS) is a key enzyme in the lipogenesis pathway that is upregulated in many cancers and has gained popularity as a druggable target of interest for cancer treatment. The first FAS inhibitor discovered, cerulenin, initially showed promise for chemotherapeutic purposes until it was observed that it had adverse side effects in mice. TVB-2640 (Denifanstat) is part of the newer generation of inhibitors. With multiple generations of FAS inhibitors being developed, it is vital to understand their distinct molecular downstream effects to elucidate potential interactions in the clinic. Here, we profile the lipidome of two different colorectal cancer (CRC) spheroids treated with a generation 1 inhibitor (cerulenin) or a generation 2 inhibitor (TVB-2640). We observe that the cerulenin causes drastic changes to the spheroid morphology as well as alterations to the lipid droplets found within CRC spheroids. TVB-2640 causes higher abundances of polyunsaturated fatty acids (PUFAs) whereas cerulenin causes a decreased abundance of PUFAs. The increase in PUFAs in TVB-2640 exposed spheroids indicates it is causing cells to die via a ferroptotic mechanism rather than a conventional apoptotic or necrotic mechanism.

ACSS2 gene variants determine kidney disease risk by controlling de novo lipogenesis in kidney tubules.

Worldwide, over 800 million people are affected by kidney disease, yet its pathogenesis remains elusive, hindering the development of novel therapeutics. In this study, we used kidney-specific expression of quantitative traits and single-nucleus open chromatin analysis to show that genetic variants linked to kidney dysfunction on chromosome 20 target the acyl-CoA synthetase short-chain family 2 (ACSS2). By generating ACSS2-KO mice, we demonstrated their protection from kidney fibrosis in multiple disease models. Our analysis of primary tubular cells revealed that ACSS2 regulated de novo lipogenesis (DNL), causing NADPH depletion and increasing ROS levels, ultimately leading to NLRP3-dependent pyroptosis. Additionally, we discovered that pharmacological inhibition or genetic ablation of fatty acid synthase safeguarded kidney cells against profibrotic gene expression and prevented kidney disease in mice. Lipid accumulation and the expression of genes related to DNL were elevated in the kidneys of patients with fibrosis. Our findings pinpoint ACSS2 as a critical kidney disease gene and reveal the role of DNL in kidney disease.

Glyceryl triacetate promotes blood–brain barrier recovery after ischemic stroke through lipogenesis-mediated IL-33 in mice

BackgroundLipid metabolism has a crucial role in neural repair in neurodegenerative diseases. We recently revealed that lipogenesis-mediated interleukin-33 (IL-33) upregulation lead to blood–brain barrier (BBB) repair after ischemic stroke. However, manipulating the key enzyme fatty acid synthase (FASN) to enhance lipogenesis was very challenging. Glyceryl triacetate (GTA) was used as a donor of acetate and precursor of acetyl coenzyme A, the key substrate for de novo lipogenesis catalyzed by FASN. Therefore, we hypothesized that GTA would promote lipogenesis the peri-infarct after ischemic stroke and contribute to the BBB repair through IL-33.MethodsMiddle cerebral artery occlusion (MCAO) was performed on C57BL mice and GTA was gavage administrated (4 g/kg) on day 2 and 4 after MCAO. Lipogenesis was evaluated by assessment of the protein level of FASN, lipid droplets, and fatty acid products through liquid chromatography-mass spectrometry in the peri-infarct area on day 3 after MCAO, respectively. BBB permeability was determined by extravasation of Evans blue, IgG and dextran, and levels of tight junction proteins in the peri-infarct area on day 7 after MCAO, respectively. Infarct size and neurological defects were assessed on day 7 after MCAO. Brain atrophy on day 30 and long-term sensorimotor abilities after MCAO were analyzed as well. The inhibitor of FASN, C75 and the virus-delivered FASN shRNA were used to evaluate the role of FASN-driven lipogenesis in GTA-improved BBB repair. Finally, the therapeutic potential of recombinant IL-33 on BBB repair and neurological recovery was evaluated.ResultsWe found that treatment with GTA increased the lipogenesis as evidenced by lipid droplets level and lauric acid content, but not the FASN protein level. Treatment with GTA increased the IL-33 level in the peri-infarct area and decreased the BBB permeability after MCAO. However, infarct size and neurological defect score were unchanged on day 7 after MCAO, while the long-term recovery of sensorimotor function and brain atrophy were improved by GTA. Inhibition of lipogenesis using C75 or FASN shRNA reversed the beneficial effect of GTA. Finally, exogenous IL-33 improved BBB repair and long-term functional recovery after stroke.ConclusionCollectively, we concluded that treatment with GTA improved the BBB repair and functional recovery after ischemic stroke, probably by the enhancement of lipogenesis and IL-33 expression.Graphical

FASN Inhibitors Enhance Bestatin-Related Tumor Cell Apoptosis Through Upregulating PEPT1.

Fatty acid synthase (FASN) is generally over-expressed in human tumor tissues and catalyzes de novo synthesis of fatty acids on which tumor cells depend. Bestatin, an inhibitor of aminopeptidase/CD13, is one of the dipeptide substrates for the human oligopeptide transporter 1 (PEPT1). In the current study, we aimed to uncover the role of FASN inhibitors in bestatininduced tumor cell apoptosis and the underlying mechanism, extending our understanding of the correlations between FASN and PEPT1 in cancer and providing a new strategy for tumor targeted treatment. Cerulenin, orlistat and siRNAs were applied to inhibit FASN. The cell viability and apoptosis were assessed with MTT (thiazolyl blue tetrazolium bromide) assays and annexin VFITC/ PI staining with flow cytometry analysis. Western blot and qRT-PCR analysis were used to detect the protein levels and mRNA levels of the indicated genes in tumor cells, respectively. Protein degradation or stability was examined with cycloheximide chase assays. CD13 activity was detected by gelatin zymography. The HT1080 and C26 xenografts models were conducted to assess the efficacy in vivo. In the current study, we found that inhibiting FASN by cerulenin and orlistat both augmented the effects of bestatin in decreasing tumor cell viability. Cerulenin increased the apoptosis rates and enhanced the cleavage of PARP caused by bestatin. Furthermore, cerulenin, orlistat and siFASNs markedly elevated PEPT1 protein levels. Indeed, cerulenin induced the upregulation of PEPT1 mRNA expression rather than affecting the protein level after the cells were treated with CHX. And Gly-Sar, a typical competitive substrate of PEPT1, could attenuate the augment of bestatin-induced cell killing by cerulenin. Moreover, synergistic restrain of tumor growth accompanied by a reduction of Ki-67 and increment of TUNEL was significantly achieved in the xenograft models. Interestingly, no clear correlation was observed between the CD13 with FASN and/or PEPT1 in tumor cells. FASN inhibitors facilitate tumor cells susceptible to bestatin-induced apoptosis involving the up-regulation of PEPT1 at the mRNA translation level and the transport of bestatin by PEPT1, emerging as a promising strategy for tumor targeted therapy.